Peptide-based immunotherapy in lupus: Where are we now?

Abstract In autoimmune rheumatic diseases, immune hyperactivity and chronic inflammation associate with immune dysregulation and the breakdown of immune self-tolerance. A continued, unresolved imbalance between effector and regulatory immune responses further exacerbates inflammation that ultimately causes tissue and organ damage. Many treatment modalities have been developed to restore the immune tolerance and immmunoregulatory balance in autoimmune rheumatic diseases, including the use of peptide-based therapeutics or the use of nanoparticles-based nanotechnology. This review summarizes the state-of-the-art therapeutic use of peptide-based therapies in autoimmune rheumatic diseases, with a specific focus on lupus.


Introduction
3][4] Despite extensive research, the precise cellular and molecular mechanisms that drive the pathogenesis of those conditions are only partly understood, with a resulting variety of therapeutic approaches that depend on the symptoms, signs, and stage of the disease.
In systemic lupus erythematosus (SLE), corticosteroids are widely used to manage inflammation including in lupus nephritis, yet they carry significant side effects. [5]Other immunosuppressive agents, including disease-modifying anti-rheumatic drugs (DMARDs) and biological therapies have shown some efficacy in inducing long-term remission but carry the risk of adverse events including the development of malignancy. [6,7]In addition, treatments with biologics can be costly and increase the risk of systemic immune suppression. [8][11] We discuss below the promise of peptide-based immunotherapies and related clinical trials.

Peptide-Based Therapeutics
Peptide-based therapeutics can be broadly categorized into two categories that are based on their chief mechanism of action: immunogenic or tolerogenic.Immunogenic peptide therapy induces an immune response against a specific antigen, whereas the tolerogenic peptide therapy induces immune tolerance to self-antigens.
[14] Although this approach has mainly been used in cancer immunotherapy to activate cytotoxic T lymphocytes (CTLs) against tumor antigens, several immunogenic peptide therapeutics have been developed as well for autoimmune diseases including SLE, [15,16] rheumatoid arthritis, [17,18] type 1 diabetes, [19,20] and multiple sclerosis, [21] with varying success.[24][25][26][27][28] This approach has been tested in several autoimmune diseases, including SLE, rheumatoid arthritis and multiple sclerosis.Tolerogenic peptide therapy can be further subcategorized according to the type of peptide(s) used, specifically: [31] They can be delivered alone or in combination with immunomodulatory agents.
(2) Whole protein peptides: These peptides are derived from full-length self-proteins and can induce a broader immune response against multiple epitopes of the protein.They can be used to induce immune tolerance or to modulate the immune response in autoimmune diseases. [13,32,33]6]

Strategies for Peptide-Based Therapy
A central characteristic of autoimmune diseases is the breakdown of self-tolerance with the activation of autoreactive immune cells that secrete pro-inflammatory cytokines and chemokines that amplify inflammation.Two different strategies have been proposed to target these key aspects of the pathogenesis of autoimmunity [37] (Table 1).
[40][41] This approach produces neutralizing antibodies (nAbs) against these proinflammatory biomolecules to inhibit their activities.In this strategy, the target immune cells are mostly B cells.
Although peptide-based therapeutic approaches have shown promising results in inducing immune tolerance in autoimmune diseases, an optimization of the design and delivery of the peptides is still crucial to improve clinical efficacy. [47]

Cytokines/Chemokines
Since pro-inflammatory cytokines and chemokines play a critical role in the pathogenesis of many autoimmune diseases, peptide-based vaccines have been designed to antagonize the pro-pathogenic cytokines and chemokines to reduce inflammation and tissue damage.
Modified peptides can target pro-inflammatory cytokines or chemokine receptors selectively.For example, a modified DNA peptide therapy targeting pro-inflammatory interleukin (IL)-17 significantly reduced organ damage and increased the survival of treated lupus-prone mice, [48] while targeting the CXC chemokine ligand 10 (CXCL10) attenuated experimental autoimmune encephalomyelitis. [49]Of interest, a peptide therapy targeting pro-inflammatory tumor necrosis factor alpha (TNF-α) induced the generation of T regs and reduced inflammation in a mouse model of collagen induced arthritis. [50,51]otential biologics have been generated to target pro-inflammatory cytokines, such as IL-1, interferon (IFN)-α, IFN-γ, IL-6, TNF-α, IL-17, and IL-21, and several clinical trials that aim at inhibiting the activities of these mediators have been initiated. [52]Despite the promising results in preclinical studies, there are still challenges that need to be addressed before translation into the clinic, including the identification of the optimal peptide targets and delivery methods, as well as the potential off-target effects and long-term safety.

Peptide-Based Tolerogenic Therapeutics in Lupus
Recent studies both in vitro and in vivo have identified several peptides that are useful in lupus treatment.Table 2 indicates effects of the current peptide therapeutics in lupus.
pCons Peptide pCons (pConsensus-FIEWNKLRFRQGLEW) is a 15-mer peptide, derived from the V H regions of anti-DNA antibodies from (NZB x NZW) F1 (BWF1) mice. [9,10,22,27,28,43,53]pCons peptide induce immune tolerance therapy in BWF1 lupus-mice by promoting the generation of autoantigen-specific regulatory T cells that suppressed effector T cells [11,54] and autoantibodyproducing B cells. [55]ons peptide therapy induces immune tolerance by promoting expansion of CD8 + and CD4 + T regs.[9,10,22,25,27,28,53,56]   pCons-induced T regs suppress the production of anti-dsDNA autoantibodies, reduce proteinuria, and delay nephritis in murine lupus. [22,53]Notably, oral administration of different forms of pCons peptide, including D and L forms, delayed anti-ds-DNA Ab production and lupus nephritis, and increased the survival of lupus mice. [57]portantly, pCons peptide induced T regs in lupus patients. [58]hen the lupus patients' peripheral blood mononuclear cells (PBMCs) were cultured with pCons peptide, the expansion of fully functional T regs occurred in those patients that were seropositive for anti-DNA autoantibodies.This is in line with the antigen-specificity of the response promoted by peptide immunotherapy, and resulted in the suppression by the pCons-induced T regs of the proliferation of CD4 + CD25 - T effectors cells and reduced production of proinflammatory cytokines. [58]Clinical trials are awaited to address whether the promising effects of pCons can be translated into clinical settings.

human complementarity-determing region 1 (hCDR1) (Edratide)
hCDR1 (edratide) is a 19-mer peptide based on the heavy chain complementarity-determining region one (CDR1) sequences of a human anti-DNA monoclonal antibody.Edratide reduced autoreactive T cells responses by inducing T regs and

cells (DCs) become aggressive and create autoimmune activation and further inflammation in SLE. B: In the tolerance state, peptide-induced tolerization promotes the expansion of antigen-specific regulatory cells and further tolerizes dendritic cells (DCs). These tolerized dendritic cells induce deletion and anergy of cognate T cells and further modulate regulatory T cells in SLE. These peptide induced regulatory T cells (T regs ) which suppress the autoreactive/autoimmune responses in SLE.
64] Earlier studies had shown that hCDR1 induced anergy in antigen presenting cells (APCs) and the generation of Tr1 T regs , which expressed tolerance-associated genes. [24,59,60]hCDR1 also downregulated IL-1β, IFN-γ, and TNF-α in addition to IFN-α, and upregulated the immunosuppressive cytokine transforming growth factor-β (TGF-β).It also reduced expression of Lymphocyte function-associated antigen 1 (LFA-1) and CD44 and regulated Jun-N terminal kinase (JNK) in the reduction of the apoptosis of T cells. [65,66]DR1 treatment delayed the production of both anti-DNA antibodies and proteinuria, and increased the survival of lupus-prone mice by regulating B cell activating factor (BAFF). [67]Additionally, hCDR1 improved cognitive behavior and brain pathology in lupus mice [68] and downregulated the expression of the indoleamine 2, 3-dioxygenase (IDO) gene, which is increased in SLE patients. [47]phase II clinical trial with hCDR1 (Edratide) was conducted in 340 SLE patients and demonstrated that it was safe and well-tolerated.However, the co-primary endpoints of a reduction of SLE disease activity index (SLEDAI-2K) and adjusted mean SLEDAI (AMS) in patients compared with controls using a landmark analysis were not met. [69]Secondary outcomes were improvement in the British Isles Lupus Assessment Group (BILAG) Responder Index and medicinal flare analysis.There was a positive trend in the Composite SLE Responder Index in the Intention-to-treat (ITT) cohort, and post-hoc analysis showed that the BILAG secondary endpoint was met for a number of subgroups dosing levels, including low or no steroids, seropositivity, and patients with 2 grade BILAG improvement.

DWEYS Peptide
[85][86] It inhibits the R4A mAb from binding to dsDNA in animal models of lupus and SLE patients.It has been shown that the nephritogenic mouse monoclonal antibody R4A binds to a consensus pentapeptide sequence D/EWD/EYS/G (DWEYS), present in both NR2A and NR2B subunits of the mouse and human N-methyl-d-aspartate receptor (NMDAR). [84,85,87]Anti-DWEYS and anti-dsDNA antibodies have cross-reactivity with NMDAR. [83]Intravenous administration of the DWEYS peptide to gestating mice had protective effects on the fetal brains when exposed to toxic doses of anti-NMDAR antibodies. [88][90] Furthermore, the peptide suppressed human autoreactive B cells and the production of anti-dsDNA antibodies in vitro. [90]SLE-412 FISLE-412 is a small molecule peptidomimetic that can neutralize anti-dsDNA autoantibodies associated with SLE.FISLE-412 has shown effectiveness in vitro and in vivo.[91][92][93] It inhibits lupus autoantibody-mediated antigen recognition by human SLE patient serum and further prevents pathogenic interactions with tissue antigens.[91] In addition, it suppresses pathogenic deposition of SLE autoantibodies into the kidney glomeruli, and blocks neurotoxicity in vivo.[91] Based on its structure-activity relationships, several analogues of FISLE-412 have been developed and found to neutralize anti-lupus antibodies in vitro and ex vivo.[92] FISLE-412 is well-tolerated and not immunogenic; as well as being stable, easily synthesized in high-throughput, and inexpensive to produce.[92,94] ALW ALW (ALWPPNLHAWVP) is a 12-mer peptide mimic identified from the four different types of murine monoclonal anti-DNA immunoglobulin G (IgG, including IgG1, IgG2a, IgG2b, and IgG3) by screening phage display libraries.[95] ALW peptide exhibits isotype-dependent binding to anti-DNA antibodies in a sequence-specific manner.In addition, it markedly inhibits the binding of anti-DNA antibodies in human and mouse lupus sera to dsDNA and to laminin self-antigen. A proteive effect of ALW peptide was demonstrated in MRL lymphoproliferation strain lupus mice, where it inhibited glomerular deposition of antibodies, reduced serum-anti-dsDNA antibodies, and improved overall renal pathology.[96] Due to the absence of amino acids such as methionine, cysteine, and glutamine, ALW is physiologically stable and is resistant to peptide oxidation, cyclization, and degradation.[97] It is nontoxic, non-immunogenic, and has a net neutral charge that reduces non-specific protein-protein interactions.As such, ALW or its analogues are seen as good potential novel candidate therapeutics in SLE.

LJP-394 (Abetimus Sodium)
LJP-394 (Abetimus sodium) is a synthetic peptide developed to induce immune tolerance to double-stranded DNA (dsD-NA), a key autoantigen in SLE. [98]It is a tetrameric oligonucleotide conjugate that can reduce anti-dsDNA antibody levels, minimize nephritic flares, [98] and act as a B cell tolerogen. [99]t has been suggested that the relative affinity of dsDNA antibodies from SLE patients for LJP-394 may impact their responses to the drug, and patients with high-affinity antibodies to LJP-394 experience a reduction in affinity after four months of weekly treatment. [100]P-394 has been evaluated in 14 clinical trials [101] and has shown to be safe and reduce circulating anti-dsDNA antibodies and disease activity in patients with active SLE.However, two pivotal trials failed to meet the primary endpoint of a statistically significant prolongation in the time to renal flare. [98,102]n another clinical trial, a dosage of 100 mg/week of LJP-394 significantly reduced anti-dsDNA antibody levels but did not significantly prolong the time to renal flare compared to placebo.Nonetheless, several positive trends in endpoints were observed in the treated group, [103] leading to wonder how to improve targeting of this agent to the most suitable SLE patients.

Use of Nanotechnology in Peptide-Induced Tolerance in SLE
[106] In SLE, nanotechnology-based strategies have been developed to induce immune tolerance to self-antigens [107] and to deliver peptides that modulate the immune response. [108]noparticles offers several advantages over traditional drug delivery systems, including improved pharmacokinetics, enhanced stability, and reduced toxicity. [105]In SLE, nanoparticles have been used to deliver lupus-specific peptides, selectively targeting autoimmune B cells, and inducing immune tolerance.For instance, poly-(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with a lupus-specific peptide (P140) have demonstrated efficacy in reducing autoantibody production and renal damage in lupus-prone mice. [109]Another study utilized T cell targeted PLGA-nanoparticles encapsulating IL-2 and TGF-β resulting in the expansion of both CD4 + and CD8 + T regs in vivo and suppression of murine lupus. [110]dditionally, the delivery of a calcium/calmodulin-dependent protein kinase-IV (CAMK4) inhibitor via nanoparticles ameliorated murine SLE. [111,112]Furthermore, microRNA125aloaded nanoparticles have also been found to restore homeostasis and ameliorate murine lupus by regulating the balance between T effector cells and T regs.[113]   Apart from delivering lupus-specific peptides, nanoparticles can be engineered to target specific immune cells and tissues.[116][117] Similarly, magnetic nanoparticles (MNPs) coated with a lupus-specific peptide have been shown to selectively target lupus B cells and induce immune tolerance. [110,118,119]Furthermore, nanotechnology-based strategies enable the delivery of immunomodulatory agents such as immunosuppressants to target specific immune cells and tissues.For instance, PLGA nanoparticles loaded with rapamycin, an immunosuppressant, have shown effectiveness in reducing autoantibody production and renal damage in lupus-prone mice. [120,121]24]

Conclusions and Future Perspectives
Peptide-based therapies for lupus have shown great promise in preclinical but clinical studies have been so far rather limited.This mandates additional clinical studies to better elucidate the potential of peptide-based therapies in SLE.
In clinical studies, peptides have demonstrated safety and tolerability; however, only variable efficacy has been demonstrated in reducing disease activity in SLE patients, possibly due to trial design that cannot preselect those patients that might benefit the most from peptide immunotherapy.Of note, peptides have nonetheless consistently shown the potential to induce immune tolerance, which is dysregulated in SLE patients.Peptide-based therapies have also shown the potential to selectively target specific immune cell populations, either B cell or T cell populations.
Peptides such as ALW and FISLE-412 have demonstrated the ability to neutralize lupus autoantibodies and prevent their pathogenic interactions with tissue antigens.More recently, the encapsulation of peptides in nanocarriers is leading to agents that can have improved pharmacokinetics, biodistribution, and bioavailability, as well as enhanced targe specificity.Nanoparticles can also help to reduce toxicity.
Despite this all, there are still challenges that need to be addressed.One is identifying the optimal peptide sequences and dosage to achieve the most effective outcomes.Another challenge is to develop the proper biomarkers to monitor response and predict treatment outcomes.One important consideration is that combination therapies that involve peptides and other immunomodulatory agents can developed but have so far not been tested.

Figure 1 .
Figure 1.The role of peptide induced immune tolerance in SLE.A: In the autoimmune state, the CD4 + and CD8 + T cells are activated, and Dendritic cells (DCs) become aggressive and create autoimmune activation and further inflammation in SLE.B: In the tolerance state, peptide-induced tolerization promotes the expansion of antigen-specific regulatory cells and further tolerizes dendritic cells (DCs).These tolerized dendritic cells induce deletion and anergy of cognate T cells and further modulate regulatory T cells in SLE.These peptide induced regulatory T cells (T regs ) which suppress the autoreactive/autoimmune responses in SLE.
To conclude, peptide-based therapies can promote immune tolerance and have shown great potential in reducing disease activity in lupus patients.With further research, new technologies such as nanotechnology and more information on how to better refine what we know for better clinical trials, peptidebased therapies could become a new avenue of treatment for SLE.